Despite intensive efforts, the immune responses induced by vaccination or natural infection fail to effectively prevent and control HIV infection. Thus, it is important to explore alternative immunization approaches and novel adjuvants to induce protective immune response that is superior to the natural immunity against HIV infection. Dendritic cells (DCs) play a critical role in the activation and maintenance of immune responses, and they are regulated by stimulatory as well as inhibitory signaling. Recently, we found that the ubiquitin-modifying enzyme A20, a negative regulator of RIG-I, TLR and TNFR signaling, play critical roles in limiting the immunostimulatory potency of APCs and the autoreactive response against self-antigens. We demonstrated that silencing of A20 drastically enhanced the stimulatory potency of TLR agonists and DC vaccines to induce both T cell and antibody responses. In this study we aim to explore and develop novel and potent adjuvants for HIV vaccines by inhibiting A20 and stimulating proinflammatory signaling cascades. The central hypothesis of this study is that 5'- triphosphate A20 siRNA (3P-siA20) that activates RIG-I and inhibits A20 can be used as a novel vaccine adjuvant to enhance anti-HIV cellular and humoral responses to higher levels that cannot be achieved by currently described vaccination approaches and may be capable of overcoming HIV's immune evasion and suppression. The specific aims for the exploratory R21 phase study are: 1. To in vitro test whether 5'-PPP-siA20 has a unique dual function of activating RIG-I and inhibiting the key negative regulator A20 of RIG-I, TLR, and TNFR signaling in DCs. 2. To test whether the bifunctional siA20 can potently stimulate DCs to induce stronger systemic and mucosal HIV-specific CTL and Th responses in mice. After completing the milestones of R21 study, we will proceed to the R33 phase study (years 3-5) to accomplish the following aims: 1. To test whether 3P-siA20 can potently stimulate DCs to more efficiently induce systemic and mucosal antibody responses against HIV Env in mice;and 2. To investigate whether in vivo immunization of 3P-siA20 as an adjuvant and HIV Env mutants with enhanced exposure of protective epitopes more efficiently induces memory T cells and neutralizing antibody responses against HIV in mice and rabbits. This study represents the first attempt of developing and testing a dual function molecule of stimulating RIG-I and inhibiting the key negative regulator of proinflammatory signaling as a novel in vivo adjuvant for HIV vaccination. This novel adjuvant could overcome the biological inhibitory barrier in APCs to allow the induction of immune responses against the weakly immunogenic, protective epitopes on HIV. PUBLIC HEALTH RELEVANCE: In this proposed study, we aim to develop a novel in vivo adjuvant capable of activating RIG-I signaling and inhibiting the key inhibitor of proinflammatory signaling for HIV vaccination to induce more potent protective cellular and humoral immune responses. The R21 phase study is intend to prove the concept that 5'triphosphate (3P) A20-siRNA (siA20) will have a unique dual function of activating RIG-I and inhibiting the key negative regulator A20 of TLR, TNFR, and RIG-I signaling in DCs. We will further prove the concept that the initiation of 3P-siA20- mediated signaling and the prolonged and enhanced proinflammatory signaling will potently stimulate DCs to induce stronger HIV-specific T cell responses in mice. After completing the milestones of R21 study, we will proceed to the R33 phase study to determine whether 3P- siA20 will potently stimulate DCs in inducing stronger HIV-specific antibody responses. We will further test whether in vivo immunization of 3P-siA20 as an adjuvant and HIV Env more efficiently induces memory T cells and neutralizing antibody responses against HIV in mice and rabbits. This study represents the first attempt of developing and testing a dual function molecule of stimulating RIG-I and inhibiting the key negative regulator of proinflammatory signaling as a novel in vivo adjuvant for HIV vaccination.